For decades, scientists have been mystified by the glow of the firefly lantern. To this day there is much debate over the physiological mechanism used to trigger the flash of the firefly. In order to understand the controlling mechanism, however, one must also understand the chemical phenomenon of chemiluminescence. Chemiluminescence is produced by an exothermic chemical reaction in which two substances react, forming an excited state. As the excited state decays, light is emitted instead of heat. The emission of light is specific to reactions in which the reactants reach an excited electronic state. From an excited electronic state the product decays and emits light, whereas in other reactions the reactants reach an excited vibrational state and emit heat as they decay. Chemiluminescence is easily confused with fluorescence, which results not from a chemical reaction, but from the absorption of different wavelengths of radiation by a reactant. Some organisms are able to carry out chemiluminescent reactions internally, in which case it is called bioluminescence5. Bioluminescence specifically involves reactants referred to as “luciferins,” which are substrates and “luciferases,” which are corresponding enzymes. “Luciferin” and “luciferase” are generic terms, which refer to a wide spectrum of substances unique to specific species2.
Fireflies use bioluminescence routinely for courtship and as a method of species and sexual identification. Scientists have been able to discern different timing patterns in bioluminescence flashes emitted from the firefly lantern. The patterns, on a basic level, are generally stereotyped and show little variability. However, it has also been discovered that minor distinctions within flash patterns occur regularly outside of a laboratory setting. This discovery has led to questions about the definable parameters of flash use as well as questions concerning the physiological execution of the flashes. It is presumed that both are related3. Scientists have generally approached these questions from two directions. The first approach is based on the idea that variability in flashes is maintained by sexual selection within the population. In other words, each flash pattern is assigned a meaning or a purpose, and variability in the flash patterns can be attributed to differences in prospective mates. The second approach, a neuroethological approach, is based on the assumption that each firefly is programmed with stereotyped flash patterns, but those flash patterns vary depending on fluctuations in the firefly’s physiological condition (i.e. state of arousal).
In order to further understand the bioluminescence of fireflies, experiments have been performed to manipulate and recreate reactions in the lantern. A key factor in determining whether the neuroethological approach is credible is to uncover the physiological mechanism that controls firefly flashing. If a trigger can be found for the flashes directly, scientists will be able to trace through reactions to address the factors that trigger the flashes on a larger scale (i.e. environmental factors, interactions with other fireflies). Studies have been done concerning the artificial production of firefly bioluminescence. In a process analogous to that of the chemiluminescence of luminol and dimethylsulfoxide, scientists Seliger and McElroy4 were able to synthetically produce a bright glow without the use of firefly luciferase. Their experiment involved the study of the effects of pH on chemiluminescent reaction4.
Two opposing proposals for control of the flash response mechanism have been presented recently. The first proposal, presented by June R. Aprille5, suggests that bioluminescence in fireflies is dependant upon oxygen as a trigger; the second suggests that hydrogen peroxide is the trigger. The oxygen-dependence proposal is based largely on the positioning of the mitochondria in the firefly light organ between the tracheolar air supply and the peroxisomes, which contain the reactants for chemiluminescence. These reactants require oxygen in order for the oxidation reaction to occur, producing the glow of the firefly. When mitochondria respirate they use the oxygen from the tracheolar air supply. This allows them to act as a gating system, keeping the bioluminescence reactants separate from the oxygen, which keep them from reacting. Nitric oxide has been proposed as the molecule responsible for opening and closing the “gate” of the mitochondria. NO, when introduced to the mitochondria, inhibits respiration, allowing oxygen to come into contact with the bioluminescence reactants. The inhibition of respiration by NO is reversible, and can be reversed by bright light. Therefore, the NO and oxygen model as a trigger suggests a method by which the flashing itself would cause the reaction to stop and start5.
The second proposal, presented by Helen Ghiradella and John T. Schmidt6, involves hydrogen peroxide instead of oxygen as the trigger for flashing based largely on the abundance of peroxisomes in the lantern. This model was created in response to the first proposal and includes a critique of the first as well as a counter proposal. The critique goes as follows: if exposure to oxygen triggers bioluminescence, a cut in the lantern that allows oxygen to enter should cause the whole lantern to light up, but experiments show that only the cut glows, and the glow is short lived. Instead, some scientists claim peroxide is a better-suited controlling agent. This proposal is still new and largely unexplored. The general idea behind the proposal is that the oxygen released by NO inhibition of mitochondrial respiration does not directly cause bioluminescence; instead it is used to make hydrogen peroxide. A sharp increase in levels of hydrogen peroxide due to the shut down of catalase (also triggered by the NO) and the introduction of oxygen would cause an explosive build-up of the compound that would in turn cause the flash to occur. Though this proposal is still somewhat speculative, speed of reaction is concurrent with the timing patterns of firefly flashes noted in other studies3. However, more research is required of the peroxisomes in the lantern, as well as the active forms and uses of oxygen that are still being discovered before the hydrogen peroxide theory can be confidently regarded6.
Due to limitations in the lab, we were unable to expand on the research and experiments described earlier. However, in order to gain a greater understanding of chemiluminescent reactions we looked to a different avenue of research exploration involving a newly pioneered instrument designed to detect nitric oxide in exhaled breath7. Measurements of nitric oxide exhaled from lungs are used to determine whether or not a person has asthma and how severe the asthma is. Higher levels of nitric oxide indicate a more severe condition. In this experiment, presented by Jill K. Robinson7, nitric oxide is detected a detection of chemiluminescence, which serves as a visual indicator or nitric oxide levels. In our experiment, described below, we demonstrated proof of the concept presented in Robinson’s7 paper. We then performed two different versions of the experiment to discover the most effective way of presenting the reaction visually.
Procedure 1
The experiment began with a proof of concept to see if NO2 gas formed from oxidized NO would luminesce when bubbled through a solution of H2O2 and luminol. Two solutions, labeled A and B respectively, were prepared. The first, to be labeled solution A, consisted of 100 mL of H2O2 (10 mL of 3% H2O2 diluted with 90 mL of water). The second, labeled solution B, consisted of .11 grams of luminol dissolved in 100 mL of water. The luminol took several minutes to dissolve completely, even with vigorous stirring. In addition, 25 mL of a bicarbonate/carbonate buffer solution was added to solution B. Next, 10 mL of solution A and 10 mL of solution B were combined in a small beaker. In a small Erlenmeyer flask copper wire was added to approximately 10 mL of HNO3. The copper and HNO3 reacted to form NO which oxidized to NO2 in the air. A stopper was added to the Erlenmeyer flask to allow the NO2 gas to accumulate. A piece of plastic tubing was run between the beaker and the flask, allowing the NO2 to reach the luminol/H2O2 solution.
Results
A brief blue flash appeared in the beaker and then quickly disappeared. The luminol/H2O2 solution turned pale yellow, possibly a result of the reaction.
Conclusion
Our hypothesis concerning the success of a chemiluminescent reaction between NO2 and a solution of equal parts luminol and H2O2 was proved correct. Two follow-up experiments were performed to determine the most effective model of presentation for our proof of concept. A brief blue flash appeared in the beaker and then quickly disappeared.
Procedure 2
First, NO2 was injected from a syringe into 100 cm of plastic tubing filled with 50 mL of luminol/H2O2 solution in an attempt to form chemiluminescent bubbles. The tubing was suspended in a vertically descending spiral using a clamp and stopped at the bottom end with a liquid release valve. The syringe was inserted into the valve and the NO2 was slowly injected to form bubbles in the solution.
Results
Except for a dim and brief glow at the fixture connecting the syringe and plastic tubing there was no significant sign of chemiluminescence.
Conclusion
We suspect that the lack of chemiluminescence in the tube is attributed to our inability to effectively keep the NO2 and luminol/H2O2 solution separated in the tube. Also, the bubbles formed seemed to be plain air bubbles as opposed to the desired bubbles of NO2 gas. Overall, this method of presentation was highly ineffective.
Procedure 3
Next, a similar experiment was performed using a test tube instead of plastic tubing. The test tube was filled with approximately 25 mL of luminol/H2O2 solution. NO2 was injected slowly from a syringe into the test tube to create bubbles.
Results
Blue chemiluminescent bubbles were formed for almost a full minute, stopping only when the syringe of NO2 gas was empty.
Conclusions
In general this procedure yielded the most visibly significant results. We hypothesize that this method was more successful due to a greater ability to control the exposure of the NO2 gas to the luminol/H2O2 solution. Through both rapid and slow injection we were able to produce a visible glow.
Table of Contents
Chemiluminescence in Fireflies
Zainab
Introduction
For decades, scientists have been mystified by the glow of the firefly lantern. To this day there is much debate over the physiological mechanism used to trigger the flash of the firefly. In order to understand the controlling mechanism, however, one must also understand the chemical phenomenon of chemiluminescence. Chemiluminescence is produced by an exothermic chemical reaction in which two substances react, forming an excited state. As the excited state decays, light is emitted instead of heat. The emission of light is specific to reactions in which the reactants reach an excited electronic state. From an excited electronic state the product decays and emits light, whereas in other reactions the reactants reach an excited vibrational state and emit heat as they decay. Chemiluminescence is easily confused with fluorescence, which results not from a chemical reaction, but from the absorption of different wavelengths of radiation by a reactant. Some organisms are able to carry out chemiluminescent reactions internally, in which case it is called bioluminescence5. Bioluminescence specifically involves reactants referred to as “luciferins,” which are substrates and “luciferases,” which are corresponding enzymes. “Luciferin” and “luciferase” are generic terms, which refer to a wide spectrum of substances unique to specific species2.
Fireflies use bioluminescence routinely for courtship and as a method of species and sexual identification. Scientists have been able to discern different timing patterns in bioluminescence flashes emitted from the firefly lantern. The patterns, on a basic level, are generally stereotyped and show little variability. However, it has also been discovered that minor distinctions within flash patterns occur regularly outside of a laboratory setting. This discovery has led to questions about the definable parameters of flash use as well as questions concerning the physiological execution of the flashes. It is presumed that both are related3. Scientists have generally approached these questions from two directions. The first approach is based on the idea that variability in flashes is maintained by sexual selection within the population. In other words, each flash pattern is assigned a meaning or a purpose, and variability in the flash patterns can be attributed to differences in prospective mates. The second approach, a neuroethological approach, is based on the assumption that each firefly is programmed with stereotyped flash patterns, but those flash patterns vary depending on fluctuations in the firefly’s physiological condition (i.e. state of arousal).
In order to further understand the bioluminescence of fireflies, experiments have been performed to manipulate and recreate reactions in the lantern. A key factor in determining whether the neuroethological approach is credible is to uncover the physiological mechanism that controls firefly flashing. If a trigger can be found for the flashes directly, scientists will be able to trace through reactions to address the factors that trigger the flashes on a larger scale (i.e. environmental factors, interactions with other fireflies). Studies have been done concerning the artificial production of firefly bioluminescence. In a process analogous to that of the chemiluminescence of luminol and dimethylsulfoxide, scientists Seliger and McElroy4 were able to synthetically produce a bright glow without the use of firefly luciferase. Their experiment involved the study of the effects of pH on chemiluminescent reaction4.
Two opposing proposals for control of the flash response mechanism have been presented recently. The first proposal, presented by June R. Aprille5, suggests that bioluminescence in fireflies is dependant upon oxygen as a trigger; the second suggests that hydrogen peroxide is the trigger. The oxygen-dependence proposal is based largely on the positioning of the mitochondria in the firefly light organ between the tracheolar air supply and the peroxisomes, which contain the reactants for chemiluminescence. These reactants require oxygen in order for the oxidation reaction to occur, producing the glow of the firefly. When mitochondria respirate they use the oxygen from the tracheolar air supply. This allows them to act as a gating system, keeping the bioluminescence reactants separate from the oxygen, which keep them from reacting. Nitric oxide has been proposed as the molecule responsible for opening and closing the “gate” of the mitochondria. NO, when introduced to the mitochondria, inhibits respiration, allowing oxygen to come into contact with the bioluminescence reactants. The inhibition of respiration by NO is reversible, and can be reversed by bright light. Therefore, the NO and oxygen model as a trigger suggests a method by which the flashing itself would cause the reaction to stop and start5.
The second proposal, presented by Helen Ghiradella and John T. Schmidt6, involves hydrogen peroxide instead of oxygen as the trigger for flashing based largely on the abundance of peroxisomes in the lantern. This model was created in response to the first proposal and includes a critique of the first as well as a counter proposal. The critique goes as follows: if exposure to oxygen triggers bioluminescence, a cut in the lantern that allows oxygen to enter should cause the whole lantern to light up, but experiments show that only the cut glows, and the glow is short lived. Instead, some scientists claim peroxide is a better-suited controlling agent. This proposal is still new and largely unexplored. The general idea behind the proposal is that the oxygen released by NO inhibition of mitochondrial respiration does not directly cause bioluminescence; instead it is used to make hydrogen peroxide. A sharp increase in levels of hydrogen peroxide due to the shut down of catalase (also triggered by the NO) and the introduction of oxygen would cause an explosive build-up of the compound that would in turn cause the flash to occur. Though this proposal is still somewhat speculative, speed of reaction is concurrent with the timing patterns of firefly flashes noted in other studies3. However, more research is required of the peroxisomes in the lantern, as well as the active forms and uses of oxygen that are still being discovered before the hydrogen peroxide theory can be confidently regarded6.
Due to limitations in the lab, we were unable to expand on the research and experiments described earlier. However, in order to gain a greater understanding of chemiluminescent reactions we looked to a different avenue of research exploration involving a newly pioneered instrument designed to detect nitric oxide in exhaled breath7. Measurements of nitric oxide exhaled from lungs are used to determine whether or not a person has asthma and how severe the asthma is. Higher levels of nitric oxide indicate a more severe condition. In this experiment, presented by Jill K. Robinson7, nitric oxide is detected a detection of chemiluminescence, which serves as a visual indicator or nitric oxide levels. In our experiment, described below, we demonstrated proof of the concept presented in Robinson’s7 paper. We then performed two different versions of the experiment to discover the most effective way of presenting the reaction visually.
Procedure 1
The experiment began with a proof of concept to see if NO2 gas formed from oxidized NO would luminesce when bubbled through a solution of H2O2 and luminol. Two solutions, labeled A and B respectively, were prepared. The first, to be labeled solution A, consisted of 100 mL of H2O2 (10 mL of 3% H2O2 diluted with 90 mL of water). The second, labeled solution B, consisted of .11 grams of luminol dissolved in 100 mL of water. The luminol took several minutes to dissolve completely, even with vigorous stirring. In addition, 25 mL of a bicarbonate/carbonate buffer solution was added to solution B. Next, 10 mL of solution A and 10 mL of solution B were combined in a small beaker. In a small Erlenmeyer flask copper wire was added to approximately 10 mL of HNO3. The copper and HNO3 reacted to form NO which oxidized to NO2 in the air. A stopper was added to the Erlenmeyer flask to allow the NO2 gas to accumulate. A piece of plastic tubing was run between the beaker and the flask, allowing the NO2 to reach the luminol/H2O2 solution.Results
A brief blue flash appeared in the beaker and then quickly disappeared. The luminol/H2O2 solution turned pale yellow, possibly a result of the reaction.Conclusion
Our hypothesis concerning the success of a chemiluminescent reaction between NO2 and a solution of equal parts luminol and H2O2 was proved correct. Two follow-up experiments were performed to determine the most effective model of presentation for our proof of concept. A brief blue flash appeared in the beaker and then quickly disappeared.Procedure 2
First, NO2 was injected from a syringe into 100 cm of plastic tubing filled with 50 mL of luminol/H2O2 solution in an attempt to form chemiluminescent bubbles. The tubing was suspended in a vertically descending spiral using a clamp and stopped at the bottom end with a liquid release valve. The syringe was inserted into the valve and the NO2 was slowly injected to form bubbles in the solution.Results
Except for a dim and brief glow at the fixture connecting the syringe and plastic tubing there was no significant sign of chemiluminescence.Conclusion
We suspect that the lack of chemiluminescence in the tube is attributed to our inability to effectively keep the NO2 and luminol/H2O2 solution separated in the tube. Also, the bubbles formed seemed to be plain air bubbles as opposed to the desired bubbles of NO2 gas. Overall, this method of presentation was highly ineffective.Procedure 3
Next, a similar experiment was performed using a test tube instead of plastic tubing. The test tube was filled with approximately 25 mL of luminol/H2O2 solution. NO2 was injected slowly from a syringe into the test tube to create bubbles.Results
Blue chemiluminescent bubbles were formed for almost a full minute, stopping only when the syringe of NO2 gas was empty.Conclusions
In general this procedure yielded the most visibly significant results. We hypothesize that this method was more successful due to a greater ability to control the exposure of the NO2 gas to the luminol/H2O2 solution. Through both rapid and slow injection we were able to produce a visible glow.References
1. "Chemiluminescence"
2. O'Kane, Dennis J., et al. "Spectral Analysis of Bioluminescence of Panellus Stypticus"
3. Carlson, Albert D., and Jonathan Copeland. "Flash Communication in Fireflies"
4. Seliger, H. H., and W. D. McElroy. "Chemiluminescence of Firefly Luciferin without Enzyme"
5. Aprille, June R., et al. "Role of Nitric Oxide and Mitochondria in Control of Firefly Flash"
6. Ghiradella, Helen, and John T. Schmidt. "Fireflies at One Hundred Plus: A New Look at Flash Control"
7. Robinson, Jill K., Mark J. Bollinger, and John W. Birks. "Luminol/H2O2 Chemiluminescence Detector for the Analysis of Nitric Oxide in Exhaled Breath"